Our goal is to determine the role of actin-myosin structural dynamics in the molecular mechanism of muscle contraction. Structures, dynamics, and interactions of myosin and actin are fundamental in understanding mechanisms of contraction and its regulation. We focus on dynamic interactions of actin with the catalytic domain of myosin, and extend this investigation to include myosin's light-chain domain and myosin binding protein-C (CPro). To gain insight into structure-functional correlation, we vary active-site ligands, protein isoforms, phosphorylation, mutation, crosslinking, and drugs. Our approach is distinguished by our emphasis on dynamics as well as structure, and on the elusive weak-binding states of actomyosin as well as the more stable strong-binding states. Our core technology is a unique combination of site-directed protein labeling, coupled with high-resolution and time-resolved spectroscopies, to test and revise detailed mechanistic models for the functional interactions of myosin and actin as they transition from weak- to strong- binding states in force generation. In Aim 1, fundamental structural dynamics of the myosin catalytic domain (CD) is investigated, using probes designed to test and revise mechanistic models of actin-activated force generation with high resolution in real time. We seek to understand temporal and spatial coordination of this complex motor. In Aim 2, allosteric changes propagated from the light chain domain (LCD) through the CD to actin will be mapped by probes, with emphasis on regulation by protein isoform and phosphorylation. Similarly, Aim 3 uses the insights of the first two aims to assess structural dynamics of the actomyosin weak and strong interactions perturbed by the presence, isoform, and phosphorylation of CPro. Feasibility (preparations, preliminary data) has been established independently for all three aims, so they are not restrictively interdependent. Instead, the three aims are synergistic, in that they share reagents and are strategically aligned to strengthen each other with new discoveries. Our 3 aims arise from ten novel hypotheses, based on findings from the previous period, converging toward a new synthesis. The assembled research team brings together a powerful combination of techniques and concepts from molecular genetics and cell culture to biophysical spectroscopy and computational simulation, to solve the molecular mechanisms of muscle contraction and regulation. This project remains grounded in fundamental biophysical mechanisms, but it is increasingly clear that the tools developed in this project can pave the way for design of molecular therapies in muscle disease. Once we understand the functions of actomyosin and its regulation, we can look to control these functions. It is anticipated that by th end of the next funding period, this project will generate the most detailed and cohesive structure-function model of muscle contraction and regulation to date, and projects will spin off involving the rational design of gene and drug therapies. More generally, the lessons learned in this project are applicable to a wide range of problems in the biophysics of muscle and molecular motors.